Construction and identification of infectious clone of S191 virus strain used for the production of live attenuated measles vaccine
10.3760/cma.j.issn.0254-5101.2014.12.006
- VernacularTitle:麻疹减毒活疫苗 S191感染性克隆的构建及鉴定
- Author:
Jian WANG
;
Xiangpeng CHEN
;
Liyuan SUN
;
Lili LI
;
Fan ZHENG
;
Jun LIU
;
Xiuyu ZHENG
- Publication Type:Journal Article
- Keywords:
Measles virus;
Reverse genetics;
RNA polymerase
- From:Chinese Journal of Microbiology and Immunology
2014;(12):921-927
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a stable infectious clone of S191 virus strain used for the pro-duction of live attenuated measles vaccine. Methods Full length cDNA of S191 strain and gene fragments encoding nucleocapsid(N),phosphoprotein(P)and RNA polymerase(L)were synthesis and respectively cloned into the vector pVAX1. The 293T cells were respectively transfected with the recombinant expression plasmids and co-cultured with Vero cells. The supernatants of cell culture were collected for identifying res-cued viruses. The indirect immunofluorescence assay was performed for virus identification. The rescued viruses at different passages in Vero cells and the sequences of the rescued viruses were analyzed. Results Restriction enzyme digestion and sequence analysis showed that the recombinant expression plasmids contai-ning the full length cDNA with an artificially engineered mutation at nucleotide 2101(C-A)and gene frag-ments encoding N,P and L proteins of S191 strain were constructed successfully. The N and P proteins were detected in Vero cells with immunofluorescence assay. A cytopathogenic effect on Vero cells was induced by rescued viruses. Conclusion The stable infectious clones of S191 virus used for the production of live at-tenuated measles vaccine were rescued successfully. An approach by using reverse genetics technique for S191 strain study was established which could be used for the development of new chimeric vaccines against measles virus.
